1. Field of the Invention
The present invention relates generally to a particle analyzing apparatus and more particularly to a method and apparatus for achieving selective, discriminate, differential classification of individual blood cell types, primarily reticulocytes utilizing light scatter-technology without the utilization of fluorescent staining techniques or materials.
2. Description of the Prior Art
Reticulocytes are defined in the medical literature as immature erythrocytes (or cells from which the nucleus has been extruded) and such cells normally account for 0.7 to 2.2 percent of the erythrocyte total count. In confirming or helping to confirm the diagnosis of diseases such as, for example, various forms of anemia or acute internal hemorrhage, the determination of reticulocytes can be of critical importance.
Microscopic examination of human blood smears on a glass slide is the universal accepted method of reticulocyte determination. This method is not only time consuming but relies on the human eye for the actual reticulocyte count. This often results in an inaccurate reticulocyte count which could also result in misdiagnosis.
One such microscopic method of determining reticulocytes is disclosed by Bjorkman, "Method for Determining Absolute Reticulocyte Count," J. Clin. & Lab. Investigation, 435-436, 1958. Therein, the described method utilizes new methylene blue to stain capillary blood and then fixes the blood cell with a diluent. The diluent fluid consists of potassium thiocyanate in dilute sulfuric acid. The fixation process is attended by an escape of hemoglobin from the blood cells. The decrease in hemoglobin content enhances the definition of the reticulum when the cell is viewed under a microscope in a blood smear on a slide.
Automated reticulocyte counting apparatus is available, but in the known instrumentation reliance is universal on the employment of fluorescence as a basis for the reticulocyte determination. Apparatus utilizing fluorescent devices are expensive, complex and require relatively costly maintenance.
It is well known that reticulocytes can be discriminated and classified by utilization of flow cytometry instrumentation when coupled with fluorescent staining. One such method involves utilizing low angle light scatter in combination with 90.degree. or high angle light scatter.
The prior art literature, scientific papers and reports, illustrate, describe, and discuss the utilization of light scatter techniques at a variety of angular positions relative to the axis of the light beam being utilized to illuminate and interrogate the sample. However, the majority of the literature material available rely on the utilization of fluorescence and fluorescent techniques and chemistry to detect reticulocytes.
Examples of such prior art teaching includes U.S. Pat. No. 4,985,174 to Kuroda et al. which describes a reagent containing a dye which intensifies the strength of reticulocyte fluorescence in a stained sample and at the same time reduces the background fluorescence of the sample, so as to raise the signal to noise ratio when fluorescence is measured. This is accomplished by a reagent containing auramine O.
U.S. Pat. No. 4,325,706 to Gershman et al. relates to a method of treating a whole blood sample with acridine orange wherein the sample is passed through an optical flow cytometry cell having a narrowed hydrodynamic focal region. Red fluorescent light and forward scattered light are detected. Based on threshold comparisons, a threshold level is developed to separate the red cells from the reticulocytes.
U.S. Pat. No. 4,883,867 to Lee et al. relates to a fluorescent composition. The utilization of thiazole orange has been found to be an effective dye for staining reticulocytes.